The Hedgehog (Hh) pathway plays a critical role in human embryogenesis and tissue differentiation, and thus, disruption of the Hh pathway is implicated in some birth defects as well as cancers. Small molecules can be used to activate or block the Hh pathway, e.g., by targeting Patched, a protein of the Hh pathway that inhibits cell division, or Smoothened, a protein of the Hh pathway that promotes cell division. For example, the alkaloid cyclopamine, which is found in plants of the Veratrum genus of the Liliaceae family, can block the Hh pathway by targeting Smoothened, thus inhibiting cell growth. Cyclopamine and other alkaloids of the Veratrum superfamily have been identified as potential therapeutic agents to treat diseases in which the Hh pathway is implicated. Likewise, biosynthetic precursors, derivatives, or synthetic derivatives of such alkaloids may be therapeutically active.
It has long been known that cyclopamine, when ingested by animals, causes severe neural defects, and now its role in tumorigenesis has garnered attention. Cyclopamine can block the action of mutated genes that produce basal cell skin carcinomas, the most common form of human cancer. Studies in mouse cells suggest that cyclopamine may be used to treat a number of cancers, including medulloblastomas in the brain and rhabdomyosarcomas in muscle. Past findings also spotlight the promise of mechanism-based treatment approaches that target specific signaling pathways that are critical to a particular cancer.
Because cyclopamine and other alkaloids of the Veratrum superfamily may prove to be useful anti-cancer drugs, there is a need for efficient production methods for these compounds. The commercial production of alkaloids and other secondary metabolites is unpredictable in that even when a plant is known to produce a particular metabolite, it is unpredictable whether the plant cells will produce the metabolite in an undifferentiated cell culture. Ma et al. has disclosed “[a]n in vitro culture system for somatic embryogenesis and green plant regeneration of Veratrum californicum,” but the Abstracts do not disclose production of alkaloids from undifferentiated cell culture. Ma et al., “Somatic Embryogenesis and Green Plant Regeneration from Veratrum californicum,” 11th IAPTC&B congress Poster Sessions P-1033. Ritala et al., “Tissue culture and genetic engineering of an important anticancer compound producing plant Veratrum californicum Duran,” Planta Medica 2006; 72. See also U.S. Pub. No. 2009/0305338. Veratrum alkaloids may also be produced in cultures of differentiated tissue such as shoots or roots. These differentiated tissues may be derived from initially undifferentiated tissue or through genetic transformation, e.g., hairy roots or shooty teratomas or the like. Transformed tissue may also be cultivated as suspension of undifferentiated cells. The present invention provides methods of producing alkaloids in undifferentiated cell culture.